Method and apparatus of separating red cells from plasma in whole blood

ABSTRACT

A method and apparatus for separating red cells from plasma in whole blood in which a diluent is added to whole blood to produce separation of the red cells. A double lumen cannula instrument may be used in withdrawing the blood from a patient and having an outer lumen for providing an anticoagulant diluent and an inner lumen for withdrawing blood plus diluent, the body portion of the instrument being approximately one inch long and made of a silastic material.

United States Patent Albisser I Oct. 2, 1973 [5 METHOD AND APPARATUS 0F3,334,018 8/1967 Smyihc ..2l()/65 SEPARA'HNG RED CELLS FROM PLASMA3,6l(),226 l0/l97l Albisser 128/2 IN WHOLE BLOOD lnventor: Anthony M.Albisser, Toronto,

Ontario, Canada Assignee: The Hospital for Sick Children,

Toronto, Ontario, Canada Filed: July 1, 1971 Appl. No.2 [$8,676

Related US. Application Data Division of Ser. No. 708,668, Feb. 27,1968, Pat. No. 3,610,266.

US. Cl. 210/532 Int. Cl A6lb 5/00 Field or Search 210/73, s3, s4, 65,

References Cited UNITED STATES PATENTS Muller 2l0/73 PrimaryExaminer-Frank A. Spear, Jr. Att0rney.lohnson, Marcus & Wray [57]ABSTRACT A method and apparatus for separating red cells from plasma inwhole blood in which a diluent is added to whole blood to produceseparation of the red cells A double lumen cannula instrument may beused in withdrawing the blood from a patient and having an outer lumenfor providing an anticoagulant diluent and an inner lumen forwithdrawing blood plus diluent, the body portion of the instrument beingapproximately one inch long and made of a silastic material.

1 Claim, 6 Drawing Figures PATENTEU 2% 3.762 567 SHEET 30F 4 ANTHow n.ALmssER INuENToR W mm A}? AGENTS METHOD AND APPARATUS OF SEPARATING REDCELLS FROM PLASMA IN WHOLE BLOOD This is a division of U.S. Pat.application Ser. No. 708,668 filed Feb. 27, 1968, now U.S. Pat. No.3,610,266.

This invention relates to a method of, and apparatus for, separating redcells from plasma in whole blood particularly of the type used inpractice and in experimental research on animals and human beings.

In hospitals and research institutions, it is often necessary towithdraw blood from an animal or a human patient over long intervals oftime and, in some tests, it may even be necessary to withdraw bloodsubstantially continuously for between 2 to hours. For example, inmetabolic testing a sugar solution is injected into a patient at aregular predetermined rate or at regular predetermined intervals whilstat the same time a continuous sampling of the patients blood is carriedout. The blood is then subjected to certain testing procedures in orderto ascertain its sugar content throughout I the period of test. For thispurpose, it is necessary to in sert a cannula into the patients vein orartery whereby the blood can be continually withdrawn. In order toprevent coagulation, it is, of course, necessary to provide ananti-coagulating diluent and therefore a double lumen cannula instrumentis normally used whereby two concentric tubes are provided, theanti-coagulant diluent flowing towards the vein or artery along theouter tube whilst the mixture of blood and anticoagulant diluent iswithdrawn through the inner tube by means of a pumping action. Stepsshould, of course, be taken to ensure that the anti-coagulant diluentdoes not enter the bloodstream of the patient.

As will be clear from the above discussion, a double lumen cannulainstrument must be provided to permit the anti-coagulant diluent to flowtowards the vein or artery and to facilitate thewithdrawal of the bloodplus diluent away from the vein or artery. The tubes from thediluent-supplying means and the blood-pumping means must be connectedeach to the respective one of the two concentric tubes forming thedouble lumen cannula. In some instances, this has been achieved byobtaining a metal two-way stop cock, welding the handle of the stop cockso that it is in an open position whereby a cannula can be passedthrough the bore thereof for use in withdrawing blood whereby one end isinserted into the vein or artery whilst the other end is connected tothe pump. A plastic sheath was fitted over that end of the cannula whichwas to project into the vein or artery and slid along the externalsurface of the cannula and over the respective part of the stop cock soas to be sealed thereto by means of a sealing compound. A sealingcompound was also sometimes used to seal the cannula to the metal stopcock so as to Accordingly, there is provided a method of separating redcells from plasma in whole blood including the steps of causing a flowof diluent and a flow of whole blood to meet and continue along a singleflow path whereby red cells separate from plasma; and providing separateflow paths for said red cells and said plasma.

From another aspect it is an object to provide apparatus for separatingred cells from plasma in whole blood.

According to this aspect there is provided apparatus for separating redcells from plasma in whole blood including a first part having twopassageways therein, a first passageway for whole blood flow and asecond passageway for the flow of diluent; said first and secondpassageways meeting to provide a common flow passageway;

More specifically there is provided apparatus also including a furtherpart into which the common flow may flow; said further part including afirst branch passageway for the passage of said red cells therealong anda second branch passageway for the passage of said plasma therealong.

Apparatus for separating red cells in blood according to the presentinvention may include a double lumen cannula instrument comprising amounting of a nonmetallic material, said mounting being formed with afirst bore extending therethrough and of such a diameter as to becapable of receiving a cannula extending through the mounting, themounting including a second bore extending from the exterior of saidmounting into a cavity at the junction of said first and second bores,

provide a fluid-tight seal. However, in practice, it was 1 said firstbore being of one diameter at one end and of a slightly greater diameterat the other end, whereby when said cannula is in position in said borewith its penetrating end protruding out of said other end, a fluid flowis possible from said second bore, into said cavity within said mountingand out through said other end to the exterior of the mounting.

An embodiment of the invention will now be described, by way of example,with reference to the accompanying drawings, in which: I

FIG. 1 is a diagrammatic representation of a double lumen cannulainstrument used in the method according to the present invention;

FIG. 2 is a diagrammatic cross-sectional view of a mould for forming theinstrument of FIG. 1;

FIG. 3 shows a rod for insertion in the mould of FIG.

2 to ensure correct formation of the double lumen can I nula instrument;

FIG. 4 is a diagrammatic representation of part of a red cell bloodseparator unit for use with a double lumen cannula instrument as shownin FIG. I or independently thereof;

FIG. 5 diagrammatically illustrates a a red cell blood separator unit;and

FIG. 6 is a diagrammatic representation of a separator system.

The double lumen cannula instrument as shown in FIG. I comprises a bodyportion 1 including an integral neck portion 2 formed as one unit, byamoulding operation, from a moulding compound, Silastic A" RTV (DowComing). The overall length of the body portion, including the neckportion, is approximately 1 inch.

During the moulding of the cannula instrument, the body portion 1 isprovided with a bore 3 of a first diameter extending for afirst distancetowards the neck .portion 2 from the opposite. end 4 of the body portion1.

further part of The cannula instrument is also provided with a secondbore 5 of a second greater diameter, the second bore extending from theregion 6 of its junction with the first bore 3 and towards the neckportion 2. The bore 5 extends through the neck portion 2 so as to forman orifice 7 to the exterior of the neck portion.

The body portion 1 is also provided with a third bore 8 extendingthrough the body portion from the exterior thereof and opening into thesecond bore 5 in the region 6. The third bore 8 is shown substantiallyat right angles to the second bore 5 although this is not, of course,essential to the invention.

In use, a 20 gauge lumen-forming cannula 9, i.e., a smaller diameterstainless steel tube, is inserted into the bore 3 with a substantialpart of its length projecting forwardly of the orifice 7 in the neckportion 2 and a part projecting backwardly from the opposite end of thebody portion 1 whereby a pumping device (not shown) can be attached bysuitable tubing to that end of the lumen-forming cannula 9. It will beappreciated that, in use, the free end of the cannula 9 is inserted intothe vein or artery of a patient whereby blood may be withdrawn forsampling. The blood vessel of a patient is diagrammatically illustratedin FIG. 1 and is identified by the numeral 10.

In FIG. 1, there is also shown a plastic outer lumen sheath ll of amedical cannula which is of such a length that its end projectsapproximately 1 mm. beyond the end of the inner lumen 9 when the two areinserted into a blood vessel 10. The plastic lumen sheath ll fits ontothe neck portion 2 of the double lumen cannula instrument and because ofthe properties of the silastic material from which the body portion ismoulded, a fluid-tight seal is achieved between the neck portion 2 andthe plastic sheath 11. It will thus be seen that an inner lumen isformed by the lumenforming cannula 9 whilst an outer lumen is formedbetween the external surface of the inner lumen-forming cannula 9 andthe inner surface of the concentric plastic lumen-forming cannula sheath11.

In use, an 18 gauge stainless steel tube 12 having a length ofapproximately three-fourth inch is inserted into the third bore 8 so asto form a fluid-tight seal therewith. Apparatus (not shown) is connectedto the stainless steel tube 12 to supply an anti-coagulant diluenttherealong and along the outer lumen formed between the tube 9 and thesheath 11 towards the blood vessel 10. Pumping means (not shown) isconnected to the end of the tube 9 remote from the blood vessel wherebyblood may be withdrawn from the blood vessel for sampling purposes. Dueto the pumping action, the anti-coagulant diluent is also drawn alongthe lumen 9 which therefore carries a mixture of blood and diluent sothat substantially no diluent is passed into the blood vessel 10.

As will be clear from the above, the double lumen cannula instrumentshown in FIG. 1 may conveniently be used in blood sampling tests on bothhuman patients and animals. Because of the particular construction ofthe lumen cannula instrument and the advantageous medical properties ofthe silastic material used, it has been found that patients find that itis not as cumbersome as the previously used metal cannula instrument andthat during a sampling period of from two to five hours the silasticcannula instrument is not as heavy and uncomfortable on the patients armas was the previously used metal instrument. Furthermore, it has beenestablished during use that the silastic cannula instrument is not asliable to form clots in the withdrawn blood as was the previously usedmetal cannula instrument. As will be clear, the double lumen cannula isparticularly useful for in vivo study of a patient and permits thecontinuous withdrawal of blood from the respective vein or artery bypermitting the simultaneous infusion of the anti-coagulant, which mixesonly with the withdrawn blood, whereby preventing the formation of clotsin the sample tube 9. The mixing of the withdrawn blood and theanti-coagulant occurs only at the tip of the inner lumen 9 which isapproximately 1 mm. inside the input end of the outer tubular sheath1 1. Thus, the anti-coagulant drawn along the inner lumen 9 does notenter the bloodstream of the patient.

The rate of removal of blood from the patient depends on the differencethe flow rates of the blood sample and the anti-coagulant diluent alongthe outer lumen. The solutions may be pumped to and from the cannula bya peristaltic pump-an auto-analyzer proportioning pump, whichestablishes a flow rate dependent on the diameter of the tubing in thepump. This ensures not only that the same volume of blood per unit timeis withdrawn from the patient but also that both flows stop if the pumpis stopped.

Due to the pressure produced by the pump, there is of course moretendency for liquid to leak out of the junctions between the plastic andmetal of the tubes 9 and 12. To avoid this, the bores 3 and 8 may bemoulded during manufacture to be slightly less than the externaldiameter of the respective tube. The bores 3 and 8 may conveniently bemoulded to 0.025 inches, 23 gauge, whilst the bore 5 may be moulded tobe 0.063 inch. The undersized bores in a plastic rubber thus ensure agood pressure fit when the stainless steel tubes are inserted and a goodplastic-to-metal seal is effected which does not leak due to the suctionof the pump.

As mentioned above, the double lumen cannula instrument according to thepresent invention may be included in a system for sampling the blood ofa patient 1 and performing a separation process thereon oralternatively, in instituting certain established test procedures. Thesequence of operations for withdrawl of whole blood in a continuousmonitoring experiment may be symbolized in block form where the doublelumen cannula instrument feeds into a block representing withdrawl whichalso has an' input from a further block labelled anti-coagulant anddiluent capable of supplying the anti-coagulant and diluent at a rate RAn output from the withdrawl block is fed to a calibration andadjustment unit at a rate R,. The calibration and adjustment unitsupplies an output at the rate (R R,) to the input of a separator unit.The rate of flow R, represents the pumping rate of a further dilution ofthe blood sample. a

The output of the separator unit will consist of two separate outputs,one being a PLASMA output and the other being a RED CELL output.

The sequential operations in the system may be considered as follows:

1. The continuous withdrawl of blood mixed in situ with ananti-coagulant and diluent.

2. The calibration and adjustment of the dilution factor D vo1ume ofwhole blood 11 :R total diluted volume R R2 3. The continuous separationof p lasma from the diluted whole blood.

4. The chemical analyses performed on both the separated plasma and thered cell suspension.

Whole blood may be continuously withdrawn from a vein or artery of theorganism by means of a double lumen cannula according to the inventionconsisting of two sterile disposable parts. The irst part may consist ofthe plastic outer lumen sheath of an Argyle Medicut cannula (ArgyleCatalogue No. AR-32l8) inserted into the blood vessel in the usualmanner. The second part of the double lumen cannula instrument mayconsist of the above-described double lumen instrument properly insertedinto the first part. In practice, the plastic outer lumen sheath ll-willbe inserted into the respective blood vessel by means of thetissue-piercing syringe which is normally available for taking a bloodsample and ensuring that one enters the respective blood vessel. Theplastic sheath normally surrounds the needle of the syringe and isinserted into the blood vessel together with the syringe needle. Afterwithdrawing the plunger of the syringe to examine the sampled blood, thesyringe needle is withdrawn whilst the plastic sheath remains insertedin the blood vessel. The lumenforming cannula 9 of FIG. 1 is then merelyinserted down the plastic sheath until it enters the blood vessel insubstitution for the aforesaid needle with the tip of the plastic sheathll projecting 1 mm. (approximately one thirty-second inch) beyond thetip of the inner lumen-forming cannula 9 within the blood vessel 10(FIG. 1). If necessary, the two lumen-forming components may be pushedfurther into the blood vessel. The pump is now started and the wholeblood sample is drawn into the cannula tip at the rate R atthe same timebeing properly mixed with isotonic diluent and anti-coagulant. Thediluent is pumped to the cannula tip along the outer lumen formed by theconcentric sheath 11 at the rate R The diluted blood is withdrawn fromthe cannula tip via the inner lumen at the rate R,, which is simply thesum of the rates R A and R,,,,,. This configuration preventsanti-coagulant and diluent from entering the bloodstream of theorganism.

In one constructed system according to the present invention, the bloodsample mixed with anti-coagulant and isotonic diluent was drawn from thedouble lumen cannula by means of a peristaltic pump (Techniconproportioning pump single speed) fitted with a manifold (Techniconmanifold) containing one pump tube (Technicon pump tubes) whose diameterdefined the flow rate R,. Tygon tubing was used to connect the cannulato the input of the pump and this should, of course, be kept as short aspossible (between two and three feet) and its internal diameter should,of course, be small about 0.025 inches. These constraints minimizelongitudinal diffusion in the tubing and also filter the flowirregularities of the peristaltic pumping. Further dilution of the bloodsample can be carried out at the peristaltic pump by mounting on thesame manifold a second tube. pumping at the rate R,,, as mentionedabove. The total dilution factor DF can be determined by measuring theflow rate R, and the combined flow rate (R, R,). From these two rates,the rate R, can be determined for any desired dilution factor byapplying the relationship R, R, DF(R, R2,

where R, is the rate of flow to the double lumen cannula tip.

Thus, the rate of whole blood removal is given by the formula Duringexperiments, it has been found that R should preferably be greater than0.1 ml/min. min. in order to minimize the peristaltically inducedfluctuations in the flow R,.

Accurate measurement of the flow rates R,'and (R, R can be simplyachieved by using two pipettes and a stop watch. The diluent at flowrate R, passes along a path through the proportioning pump rollers to athree-way stop cock capable of passing the diluent either to waste orinto the flow line of the flow at rate R, (blood plus anti-coagulantdiluent), after the respective proportioning pump roller in thedirection of flow. When the first-mentioned stop cock is in the correctposition, the combined flow (R, R,) is passed to a further three-waystop cock inserted in the fluid line whereby the fluid flow may bearranged to enter a 5 ml. pipette.

To measure R,, the flow R is diverted to waste by the firstmentionedstop cock and the flow R, is directed into a 2 ml. pipette by means ofthe second-mentioned stop cock. The time to pump a known volume ofliquid is measured and the flow can then be calculated. For

I the flow (R, R as mentioned, a 5 ml. pipette is used and by this meansboth flows can be measured.

In FIG. 2, there is shown a mould for manufacturing the silastic cannulainstrument of FIG. 1. The mould may conveniently be a two-piece mouldmade of the plastic referred to as Lucite (registered trade mark). Thismould is shown in cross section in FIG. 2 and it will be appreciatedthat prior to drilling and milling the mould, two pieces of Lucite arefitted together and held by means of three thirty-second inch studsapproximately 1 inch long and having a half-round end together with athreaded opposite end. Conveniently, Lucite blocksmay be used having alength several times the size of the required cannula instrument and,for example, a Lucite block may be 4-% inches long so that six mouldsmay be drilled and milled into each block so as to produce the bodyportions for six double lumen cannula instruments according to thepresent invention. Thus, two pieces of Lucite material would be requiredhaving dimensions seven-sixteenths X 1-1/4 X 4-% inches. The dimensionsrequired for each mould are indicated in FIG. 2 for convenience, theoverall ond bore 5 of the double lumen cannula instrument of FIG. 1. Thewall of the mould 20 is provided with a drilled, or otherwise formed,bore 24 having a diameter of 0.025 inch and emerging into the main space21 of mould 20.

' In FIG. 3, there is shown a rod 25 for insertion within the mould ofFIG. 2 to ensure that the bores 3, 5 and 8 (FIG. 1) are properly formed.The rod 25 is of onesixteenth inch diameter and 1 inch total length. Atone end it is provided with an axially located bore 26 and a transversebore 27 as shown in FIG. 3.

During a moulding operation, the end 28 of the rod 25 is inserted in thespace 23 (FIG. 2) with the major portion of the rod 25 projecting intothe space 21. To ensure that the bores 3 and 8 are properly formed, twopieces of wire, such as piano wire, are used. The first piece of wirehaving a diameter of 0.024 inch and a length of approximately 56 inch isinserted in the bore 26 of rod 25 so as to project upwardly through thetop of the mould 20. The second piece of wire having a diameter of 0.024inch and a length of approximately 3/4 inch is inserted in the bore 27so as to project horizontally through the bore 24 in the mould 20 ofFIG. 2. Thus, when the silastic A RTV (Dow Corning) moulding compound isinjected into the mould, the bores 3, 5 and 8 will be properly formed.

The steps in the moulding operation may be summarized as below.

1. Ensure that the components of the mould are clean and dry.

2. Assemble the Lucite pieces of the mould together using, for example,three thirty-seconds inch diameter threaded studs approximately 1-56inch long.

3. Insert the rod or wire into the axially located bore 26 of rod 25.

4. Clamp the assembled combination of rod 25 and the abovementioned rodor wire with a pair of hemostats and insert the end 28 of rod 25 intothe It; inch diameter hole 23 in the LUcite mould.

5. Align the bore 27 in rod 25 with the 0.025 inch diameter bore 24 inthe mould 20 of FIG. 2.

6. Push the end of the respective rod or wire through the 0.025 inchbore 24 in mould 20 and into the bore 27 of rod 25 (FIG. 3).

7. Prepare the moulding compound, silastic A RTV (Dow Corning) mouldingcompound, and inject it into the mould, starting at at the bottom andfilling the mould to the top so as to ensure that no air bubbles aretrapped by the silastic material.

After. the silastic material has properly set and catalyzed, the mouldmay be taken apart as follows.

1. Remove the rod or wire from the bore 27 by merely pulling it straightout.

2. Remove the threaded bolts or studs holding the two parts of theLucite mould together.

3. Carefully separate the two parts of the Lucite mould.

4. Remove the silastic double lumen attachment from the Lucite mould.

5. Carefully pull out the remaining rods or wires, i.e., the rod 25 andthe wire previously inserted into the bore 26 thereof.

6. Insert the two pieces of stainless steel tubing 9 and 12 of FIG. 1into the moulded silastic body portion.

The stainless steel tubing constituting the lumenforming cannula 9 andthe stainless steel tube 12 of FIG. 1 must be carefully checked toensure that no burrs, fragments of steel, or dirt are introduced intothe final instrument. For the instrument of FIG. 1, the stainlesssteeltubing 9 should be of 20 gauge and approximately 4 inches long and, instep 6, is passed axially through the moulded body portion. The tube 12should be of 18 gauge stainless steel tubing and approximately 56 inchlong and, in step 6, would be inserted perpendicularly into the mouldedbody portion. In this way, a double lumen cannula instrument as shown inFIG. 1 would be constructed.

Sterilization of the double lumen cannula instrument may be effected byany of the usual methods.

The double lumen cannula instrument described above may conveniently beused for in vivo monitoring of blood parameters in response to drugsand/or other substances in humans or animals. General cannulationsinvolving a double lumen catheter of any length can employ the doublelumen cannula instrument to provide the anti-coagulant to the site ofblood removal. Provided that the blood vessel is found and enteredquickly, the procedure in using the described double lumen cannulainstrument is simple and straight forward, taking only a minute or so,and once the pump is started the blood sample may be withdrawncontinuously for as long as necessary and advisable. The double lumencannula instrument has proved, in use, to be both rigid and strong andit has been discovered by experimentation that practical cannulainstruments constructed of the above-mentioned silastic rubber mouldingcompound are particularly useful for medical purposes. Furthermore, thedescribed double lumen cannula instrument may be regarded as adisposable item, for use in one operation only.

Turning now to another aspect, I have designed apparatus for separatingblood by a new method.

In medical practice and research on animals and humans, it is sometimesnecessary to separate whole blood into its constituentcomponents.-Centrifugation methods have previously been used inseparators to separate the respective components from diluted wholeblood. As is known, some chemical analysis must be made on plasma ratherthan on whole blood and therefore separated flows, one of diluted plasmaand the other of diluted plasma plus the cells, are obtained byseparation techniques so that the appropriate chemical analysis may bemade.

By pumping a whole bloodstream into a diluent stream so dilution occurscontinuously, hence dynamically (dynamic dilution), whole blood may beseparated into two laminar streams. The upper stream will consist mainlyof diluent and plasma whilst the lower stream will consist ofconcentrated red cells and some plasma. In other words, the settlingtime of red cells can be greatly enhanced when a small flow .of blood ispumped into another flow of diluent. Settling then occurs in about onesecond and the liquid proceeds in two layers along the tubing of theapparatus used.

In FIG. 4, there is shown a part of a red cell blood separator unit.

The unit shown in FIG. 4 comprises a first part 30 having across-sectional shape as shown so as to form two passageways, a firstpassageway 31 for the flow of diluent and a second passageway 32 for theflow of whole blood. The passageway 32 joins the passageway 31 at rightangles thereto and the output flow of the first part 30 continues in asingle flow path along an outlet passageway 33 in-line with thepassageway 31. A passageway 34 of a nipple part 35 is provided in-linewith the passageway 31 the part 35 beingheld in abutting relationshipwith the part 30 by means of a Tygon (trade mark name) sleeve 36. Alength of Tygon tubing 37 is provided on the end of the nipple part 35whereby the flow thereto may be fed to subsequent apparatus.

The first part 30 may conveniently be a glass T fitting type D0 or D1.(Technicon) It is important to note that air segmentation of the streamsis not used because the air bubble tends to remix the blood cells andthe plasma. Separators are effective to remove the lower layer of redcells. In FIGS. 5, there is shown a further part of a red cell bloodseparator unit whereby the concentrated red cells and some plasma may beseparated from the partially cell free diluted plasma. The incominglaminar flow of diluted plasma and settled red blood cells travels alonga length of diluted plasma and settled red blood cells travels along alength of tubing 38, through a nipple part 39 and through an in-linepassageway 40 of a further part 41. The nipple part 39 and the furtherpart 41 are held in abutting relationship by means of a sleeve 42.

The combined flow of diluted plasma and settled blood cells travelsalong the passageway 40 into a junction region 43 where the concentratedred cells travel along a lower branch passageway 44 whilst the partiallycell free diluted plasma travels along an upper branch passageway 45.The concentrated red cells travelling along the lower passageway 44will, in fact, include some plasma therein but by using three cascadedseparator units I have been able to separate in excess of 99 percent ofthe red cells. This figure is, of course, subject to rechecking.

The partially cell free plasma flowing along passageway 45 may, ofcourse, be passed through a second and then a third separator to furtheraccomplish separation. I believe that the first separator will removealong passageway 40 a volume of fluid corresponding to about twiceexpected red cell volume. Subsequent separators do approximately thesame, but the net flow is less because the red cell concentration hasalready been reduced by the first separator unit.

For proper settling of the whole blood, the tubing connecting eachseparator unit should be preferably maintained horizontal and the locusof its path should be kept smooth with only gradual changes. Smoothtransition along the passageways formed by the respective tubingsinternal diameters and the glass fittings must be maintained to avoidturbulence and nonlaminar flow which results in mixing of the twostreams.

A slight downward slope of the whole apparatus should enablethe redcells to slide and flow at thc same velocity as the supernatant plasmaand diluent. 1

Obviously, completeness of separation and volume of cell free plasmabear to each other a reciprocal relationship.

At the present moment, I do not have a concrete theroretical explanationof the settling phenomenon. It does, however, occur in solutions at roomtemperatures as well as in ice-water and with diluents as different as0.15 molar NaCl and 0.15 M LiNO,. In experiments, isotonic lithiumnitrate has been used as a diluent because the lithium ion acts as aninternal reference in-the flame photometry of sodium and potassium.

It is expected that the flow pattern of the red cells in the small boretubing and the sudden encounter with a new environment, namely that ofthe diluent, causes a change whereby the cells drop out of suspension.To contrast this effect, a well-agitated prediluted sample of bloodsettles slowly, very slowly compared to the settling of the cells whendiluted dynamically, i.e., with both solutions in motion. Furtherexperiments should be carried out to appreciate the hydrodynamicproperties of these diluted suspensions of red blood cells.

channel measures glucose in the separated red cell suspension.

The advantages of the continuous separation technique described aboveare believed to be as follows:

1. The cells are separated anaerobically, rapidly, and continuously.

2. The separation occurs one line as it were.

3. The gravitational stresses applied-to the cells are insignificant.

4. The cells are not packed tightly together so that there is lesschance of their contents exchanging with their environment.

5. The separated plasma-is removed from the cells almost immediately.

The system also introduces several advantages:

6. The dilution factor can be easily controlled by adjusting R 7. Theflow rate R of blood withdrawn can be made very small, 0.01 ml/min. sothat frequent measurements may be made.

8. The system is particularly useful in continuous monitoring workbecause it eliminates the need for discrete batch centrifugation andthereby permits complete automation and integration of thewithdrawalanalysis system.

There are also some possible disadvantages:

1. The measured plasma concentrations depend on the sample hematocritbecause the whole blood is diluted first and then separated;in contrastto first separating the plasma from the cells and then diluting theplasma for analysis. The latter method is believed to introduce nohematocrit dependence.

The described separator method and apparatus may be used in conjunctionwith the double lumen cannula instrument illustrated in FIG. 1 or,alternatively, it may be used separately'therefrom or with othersuitable apparatus.

I have constructed a multiple separation unit for con-' proportioningpump is utilized to ensure continuous blood withdrawal. The pump tubeshoulder colours areindicated on the respective lines.

Identification of the respective parts in FIG. 6 is indicated in thefollowing list.

A-Tygon tubing 0.065"- ID.; Length, 4 ft. minimum BTygon tubing 0.065"ID.; Length, 8 in. minimum C-Tygon tubing 0.065" ID.; Length, 8 inminimum D-Tygon tubing 0.025" ID.; Length, any E-Tygon tubing variesI.D.

F-Tygon tubing varies I.D. GTygon tubing varies LD.

Lengths, adjusted to properly phase flows from The interconnecting tubesA, B and C serve the additional purposes of maintaining laminar flow aswell as allowing settling of the red cells. It is necessary that they beplaced to avoid any sharp bends or twists which would cause mixing ofthe two ribbons of fluid in the tubes.

All connections should be made properly using the appropriate nipples sothat laminar flow is maintained throughout.

The red cell collecting tubes should be adjusted in length and internaldiameter so that the three flows are in phase at the summation point Z.In this way, an analysis, such as glucose, performed on the red cellsuspension will not suffer from excessive mlxing or loss of response toa step change in concentration.

To maintain the response of the separators it is also advantageous toplace the separators in a descending cascade allowing the more viscousred cells to slide down a ramp thereby maintaining equal the velocity ofboth separated streams.

For different conditions, different pump tubes may be used. Perhaps onlytwo instead of three separators need be used. The configuration shown isnot necessarily the best; it removes better than 99 percent of the redcells, but proper adjustment of the orientation and settling lengths canincrease this figure.

I claim:

1. Apparatus for separating red cells from plasma in whole bloodincluding:

a. a first part having two passageways therein, a first passageway forwhole blood flow and a second passageway forthe flow of diluent, saidfirst and second passageways meeting to provide a common flowpassageway;

b. a further part into which the common flow may flow, said further partincluding a first branch passageway for the passage of said red cellstherealong and a second branch passageway for the passage of said plasmatherealong; and

c. double lumen cannula instrument comprising:

i. a body portion moulded from silastic;

ii. said body portion including an integral neck portion of silastic andof smaller cross-sectional area than said body portion;

iii. a first bore of a first diameter extending for a first distancetowards said neck portion from the opposite end of said body and openinginto a second bore;

iv. said second bore being of a second greater diameter than said firstbore and extending from its junction with the first bore towards andthrough said neck portion to form an orifice to the exterior of saidneck portion;

v. an inner-lumen-forming cannula extending through said first andsecond bores;

vi. said first diameter being substantially equal to the externaldiameter of the inner-lumen-forming cannula so as to provide afluid-tight seal between said body portion and the inner-lumenformingcannula;

vii. a third bore extending through said body portion from the exteriorthereof and opening into said second bore substantially in the region ofsaid junction of said first bore and a tubular member in said third borehaving one end emerging into said region and the other end extending tosaid exterior of the body portion;

viii. a plastic cannula sheath concentric with said inner cannula andhaving one end fitting over said neck portion whereby an outer lumen isformed between the external surface of said inner-lumen-forming cannulaand the inner surface of said plastic cannula sheath;

ix. whereby, in use during the sampling of a patient's blood ananti-coagulant diluent can be passed along said outer lumen to the outertip of the inner lumen and blood plus anti-coagulant diluent can bewithdrawn along said inner lumen.

